Small proteins are effective affinity ligands for in vivo molecular targeting, both for molecular imaging and targeted therapy. Though there are multiple reports of charge impacting tumor delivery and background retention, systematic studies have not been performed to provide clear understanding of the correlations between charge - net, number, and distribution - and physiological performance. We will fill this gap in knowledge by in vivo study of a systematic set of protein mutants. We have engineered an 11 kDa fibronectin type III domain with high affinity for epidermal growth factor receptor (EGFR) and validated its use for molecular positron emission tomography (PET) of EGFR-positive tumors. We aim to implement designed charge mutations in this lead agent, as well as an alternative affibody ligand, to engineer EGFR molecular PET imaging agents with enhanced tumor targeting and reduced background. We will use PET and excised tissue gamma counting with tumor xenografted mice to quantify tumor targeting and background uptake and clearance. We will also evaluate the relative efficacy of theoretical and evolutionary approaches for engineering charge within proteins. Three specific aims will be employed: (1) Modulate ligand charge - net charge, number of charges, and distribution - to optimize the balance between extravasation and clearance to elevate tumor targeting magnitude and specificity. (2) Reduce renal retention by modulating net charge, number of charges, and charge distribution. (3) Efficiently modify ligand charge distributions without hindering solubility, stability, affinity, and production via a hybrid of natural sequence frequency analysis, computation of stability, and evolutionary approaches. The result of this research will be ligands with improved biodistribution that elevate the sensitivity and potency of EGFR molecular imaging and targeted therapy while reducing background signal and toxicity. Moreover, we will validate and facilitate inclusion of molecular charge as a standard design parameter in ligand engineering.